Antiphospholipid (aPL) antibodies is the term generally given to describe autoantibodies that are associated with thrombosis, recurrent fetal loss and thrombocytopenia as the primary anti-phospholipid syndrome (APS) as well as autoimmune diseases such as systemic lupus erythematosus (SLE). Harris et al. (1983) Lancet 2:1211-1214; and Lockshin et al. (1985) N. Engl. J. Med. 313:152-156. APS may be primary, or secondary, meaning that it is associated with other conditions, primarily SLE. PHOSPHOLIPID-BINDING ANTIBODIES (Harris et al., eds., CRC Press, Boca Raton, Fla., 1991; McNeil et al. ADVANCES IN IMMUNOLOGY, Vol. 49, pp. 193-281 (Austen el al., eds., Academic Press, San Diego, Calif., 1991)). aPL antibodies include so-called anticardiolipin (aCL) autoantibodies, which are discussed below. aPL antibodies (including aCL antibodies) are detected in many conditions but only the β2GPI-dependent antiphospholipid antibodies found in association with autoimmune disease require the presence of the phospholipid binding serum protein, β2GPI. Vaarala et al. (1986) Clin. Immunol. Immunopathol. 41:8-15.
Approximately 30% of patients possessing persistent aPL antibodies have 1<suffered a thrombic event. The presence of aPL antibodies defines a group of patients within SLE who display a syndrome of clinical features consisting of one or more of thrombosis, thrombocytopenia (TCP), and fetal loss. The risk of this syndrome in SLE overall is around 25%; this risk increases to 40% in the presence of aPL antibodies and decreases to 15% in their absence. Because aPL antibodies were thought to be directed at phospholipids in plasma membranes, it has been postulated that they may exert direct pathogenic effects in vivo by interfering with hemostatic processes that take place on the phospholipid membranes of cells such as platelets or endothelium. In patients with APS, the fact that aPL (including aCL) antibodies appear to be the only risk factor present is further evidence that these antibodies have a direct pathogenic role. Induction of APS by passive transfer of mice with human aPL antibodies is the best evidence yet that aPL antibodies are directly pathogenic. Bakimer et al. (1992) J. Clin. Invest. 89:1558-1563; Blank et al. (I 991) Proc. Natl. Acad. Sci. 88:3069-3073. Estimates vary but in about 15% of all stroke patients, aPL antibodies are thought to be an important contributing factor.
The clear correlation between presence of these antibodies with a number of disorders compels their detection and measurement. However, measurement of aPL antibodies in the clinical environment is still an imperfect art and therefore presents significant problems. A commercially available set of standard antisera (APL Diagnostics, Inc., Louisville, Ky.) allow generation of a standard curve for comparison of assays performed in various laboratories. A great deal of inconsistency exists, however, between the results obtained at these laboratories regarding the exact GPL and MPL, the unit of measurement for IgG and IgM antiphospholipid antibodies, respectively, ratings for given sera and the levels of GPL and MPL that are categorized as high (80 or greater), medium (20-80), low (10-20) or normal (0-10). The available commercial kits vary greatly in the values assigned to the commercially available standards. Reber et al. (1995) Thrombosis and Haemostat. 73:444-452.
The exact nature of the antigenic specificity of aPL autoantibodies is controversial, and is reflected in the evolving nomenclatures used for these antibodies. At first these autoantibodies were thought to be directed against anionic phospholipids, hence the name “anticardiolipin antibodies”. Gharavi et al. (1987) Ann. Rheum. Dis. 46 m: 1-6. It then became apparent that β2GPI played an important role in the antigenic specificity of aPL (including aCL) antibodies. Vermylen et al (1992) J. Lab. Clin. Med. 120:10; McNeil et al. (1990) Proc. Natl. Acad Sci. USA 87:41204124. These observations indicate that these antibodies are more properly called “β2GPI-dependent antiphospholipid autoantibodies”, which is the term used in this specification.
The reports that β2GPI played a role, as a cofactor, in the binding of β2GPI-Us dependent antiphospholipid antibody coupled with some reports that β2GPI-dependent antiphospholipid antibodies could bind β2GPI itself has led to conflicting interpretations as to the nature of the antigenic site recognized by these antibodies. However, the role β2GPI played has remained unclear, and several explanations have been suggested. Some groups have concluded that β2GPI-dependent antiphospholipid antibodies recognize a complex antigen that includes both β2GPI and anionic phospholipid, whereas others have observed β2GPI-dependent antiphospholipid binding to β2GPI in the absence of phospholipid. McNeil et al. (1990) Proc. Natl. Acad Sci. USA 87:4120-4124; Galli et al. (1990) Lancet 335:1544; Roubey et al (1995) J. Immun. 154(2): 954-960; Arvieux et al. (1991) J. Immunol. Methods 143:223. A number of explanations have been offered to explain these differences. Galli et al. postulate that because β2GPI dependent antiphospholipid antibodies are low affinity antibodies to β2GPI they require engagement of both combining sites on a given IgG molecule by a multivalent solid phase antigen. Galli et al. (1990). They further argue that under certain conditions, for example gamma irradiation of microtiter wells, that sufficient β2GPI can be immobilized to allow for these low affinity antibodies to bind. Others argue that a cryptic epitope, recognized by β2GPI-dependent antiphospholipid antibodies, is generated when β2GPI binds to either gamma irradiated well or to wells coated with cardiolipin. Matsuura et al. (1994) J. Exp. Med. 179:457.
β2GPI is a 50 kilodalton plasma glycoprotein that displays several properties ° defining an anti-coagulant, such as inhibition of contact activation of the intrinsic coagulation pathway, platelet prothrombinase activity, and ADP-induced platelet activation. Roubey (1996) Arthritis Rheum. 39:1444; Valesinit et al. (1992) Automimmunity 14:105. The amino acid sequence of β2GPI has been determined. Lozier et al. (1984) Proc. Natl. Acad. Sci. USA 81:3640; Steinkasserer et al. (1991) Biochem. J. 277:387. β2GPI is composed of five homologous domains. Four of them are composed of approximately 60 amino acids that contain highly conserved cystines, prolines and tryptophans. Lozier et al. (1984) Proc. Natl. Acad Sci. USA 81:3640; Steinkasserer et al. (1991) Biochem. J. 277:387-391. This protein structural motif was first described in β2GPI and is characterized by its length, independent folding, and by a framework with the homologous location of four half-cystine residues involved in the formation of two internal disulfide bridges; two prolines; two phenylalanine, tyrosine or histidine residues; two glycines; and one leucine or valine.
These repeating motifs were designated as sushi structures because of their shape or are sometimes referred to as short consensus repeats. Reid et al. (1989) Immunol. Today 10:177; Ichinose et al. (1990) J. Biol. Chem. 265:13411-14. The fifth domain contains 82 amino acid residues and 6 half-cystines.
In addition to the above-discussed controversy surrounding the nature of the antigenic specificity of β2GPI-dependent antiphospholipid antibodies, there has been considerable controversy regarding the nature and location of epitopes recognized by β2GPI-dependent antiphospholipid antibodies in β2GPI. It has been suggested that the phospholipid-binding site of β2GPI is located in the fifth domain. Hunt et al. (1993) Proc. Natl. Acad. Sci. USA 90:2141. Hunt et al. also reported on the structural differences between an active form of β2GPI and an inactive form of β2GPI that lacked β2GPI-dependent antiphospholipid cofactor activity and concluded that the putative epitope for β2GPI-dependent antiphospholipid antibodies was most likely to be in the fifth domain of β2GPI. Hunt et al. (1994) J. Immunol. 152:653-659. Other groups have used recombinant β2GPI proteins to attempt to locate the antigenic site of 2GPI-dependent antiphospholipid antibodies. Two of these groups produced β2GPI mutant proteins from which various domains had been deleted in a baculovirus expression system. Both groups concluded that the epitope for β2GPI-dependent antiphospholipid antibodies was cryptic and that domain 4 may be dominantly involved in the exposure of the epitope. Igarashi et al. (1996) Blood 87:3262-3270; George et al. (1998) J. Immunol. 160:3917-3923. Another group expressed β2GPI mutant proteins from which various domains had been deleted in Escherichia coli and concluded that domain 5 contained epitopes recognized by β2GPI-dependent antiphospholipid antibodies. Yang et al. (1997) APLAR J. Rheumatol. 1:96-100.
There is a serious need for improved detection systems and toleragens for β2GPI-dependent antiphospholipid antibody-mediated conditions.
All references cited herein are incorporated by reference in their entirety.